Alzheimer's disease (AD), the most common dementia in the elderly, poses a major public health problem. There is an urgent need to further our understanding of AD pathogenesis and to identify molecular mediators as novel therapeutic targets for treating this debilitating disease. Activation of the brain's microglial cells and subsequent inflammatory processes are important in the progression of AD. We have shown that cathepsin B (CatB) released from Abeta-stimulated microglial cells plays a critical role in microglial-mediated neuronal damage in vitro. Catalytically active CatB has been observed extracellularly in association with neuritic plaques in AD. Degradation of extracellular matrix proteins and associated signal transduction molecules by CatB can trigger apoptosis and potentially contribute to neuronal loss in AD. Specific Aim 1 will extend our in vitro findings and explore the role of CatB in AD pathogenesis in vivo. By crossing CatB knockout mice with transgenic mice overexpressing hAPP carrying FAD-linked mutations directed by the PDGF beta-chain promoter (APP-FAD), we will examine whether genetic inactivation of CatB protects against neuronal and synaptic deficits in APP-FAD mice. This study will also allow us to evaluate CatB as a potential therapeutic target for AD. Cystatin C (cysC), the most potent endogenous inhibitor of CatB has been implicated in neurodegenerative diseases. The polymorphism in CTS3, the gene encoding cysC, is a potential risk factor for sporadic late-onset AD. Our pilot study revealed a significant decrease of cysC levels in young APP-FAD mice in association with neuronal deficits in both cortical and hippocampal subregions. We hypothesize that cysC plays a protective role in AD pathogenesis by counteracting the neurotoxic effects of CatB. Specific Aim 2 will examine the effects of cysC on the neuronal deficits in APP-FAD mice. First, a comprehensive analysis of cysC expression in APP-FAD mice of different age groups will correlate cysC levels with different stages of neuronal and synaptic degeneration in APP-FAD mice. Second, using a lentiviral vector-based gene delivery system, we plan to elevate cysC levels in selected brain regions of the APP-FAD mice and to examine the protective effects of cysC against neurodegeneration. These studies will shed light on the potential role of cysC in AD pathogenesis and further our understanding on the CatB-mediated neuronal damage in AD.